Molecular mechanisms of double strand break repair in eukaryotes

Genome stability is of primary importance for the survival and for the proper functioning of all organisms. Double strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults. In response to even a single DSB, organisms must trigger series of events to promote repair of the DNA damage in order to survive and restore chromosome integrity. In doing so, cells must regulate a fine balance between potentially competing DSB repair pathways. Much of what we know today on the mechanisms of repair in eukaryotes come from studies carried out in budding yeast. In this review, the main attention is focused on exciting new work eminating from yeast research that provides fresh insights into the DSB repair process.     

Involvement of Nucleotide Excision and Mismatch Repair Mechanisms in Double Strand Break Repair

Living organisms are constantly threatened by environmental DNA-damaging agents, including UV and ionizing radiation (IR). Repair of various forms of DNA damage caused by IR is normally thought to follow lesion-specific repair pathways with distinct enzymatic machinery. DNA double strand break is one of the most serious kinds of damage induced by IR, which is repaired through double strand break (DSB) repair mechanisms, including homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent studies have presented increasing evidence that various DNA repair pathways are not separated, but well interlinked. It has been suggested that non-DSB repair mechanisms, such as Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and cell cycle regulation, are highly involved in DSB repairs. These findings revealed previously unrecognized roles of various non-DSB repair genes and indicated that a successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. One of our recent studies found that suppressed expression of non-DSB repair genes, such as XPA, RPA and MLH1, influenced the yield of IR induced micronuclei formation and/or chromosome aberrations, suggesting that these genes are highly involved in DSB repair and DSB-related cell cycle arrest, which reveals new roles for these gene products in the DNA repair network. In this review, we summarize current progress on the function of non-DSB repair-related proteins, especially those that participate in NER and MMR pathways, and their influence on DSB repair. In addition, we present our developing view that the DSB repair mechanisms are more complex and are regulated by not only the well known HR/NHEJ pathways, but also a systematically coordinated cellular network.Key Words: Ionizing radiation (IR), DNA damage, DSB repair, NER, MMR and cell cycle.     

Mammalian X-ray-sensitive mutants which are defective in non-homologous (illegitimate) DNA double-strand break repair

In all organisms multiple pathways to repair DNA double-strand breaks (DSB) have been identified. In mammalian cells DSB are repaired by two distinct pathways, homologous and non-homologous (illegitimate) recombination. X-ray-sensitive mutants have provided a tool for the identification and understanding of the illegitimate recombination pathway in mammalian cells. Two (sub-)pathways can be distinguished, the first mediated by DNA-PK-dependent protein kinase (DNA-PK), and the second directed by the hMre11/hRad50 complex. A variety of mutants impaired in DSB repair by illegitimate recombination, with mutations in Ku, DNA-PKcs, XRCC4 or nibrin, have been described. Herein, the characterization of these mutants with respect to the impaired cellular function and the molecular defect is provided. Further studies on these mutants, as well as on new mutants impaired in as-of-yet unidentified pathways, should be helpful to a better understanding of DSB repair and of the processes leading to genome instability and cancer.     

Differential usage of non-homologous end-joining and homologous recombination in double strand break repair

Repair of DNA double strand breaks (DSBs) plays a critical role in the maintenance of the genome. DSB arise frequently as a consequence of replication fork stalling and also due to the attack of exogenous agents. Repair of broken DNA is essential for survival. Two major pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to deal with these lesions, and are conserved from yeast to vertebrates. Despite the conservation of these pathways, their relative contribution to DSB repair varies greatly between these two species. HR plays a dominant role in any DSB repair in yeast, whereas NHEJ significantly contributes to DSB repair in vertebrates. This active NHEJ requires a regulatory mechanism to choose HR or NHEJ in vertebrate cells. In this review, we illustrate how HR and NHEJ are differentially regulated depending on the phase of cell cycle and on the nature of the DSB.     

Playing the end game: DNA double-strand break repair pathway choice

DNA double-strand breaks (DSBs) are highly toxic lesions that can drive genetic instability. To preserve genome integrity, organisms have evolved several DSB repair mechanisms, of which nonhomologous end-joining (NHEJ) and homologous recombination (HR) represent the two most prominent. It has recently become apparent that multiple layers of regulation exist to ensure these repair pathways are accurate and restricted to the appropriate cellular contexts. Such regulation is crucial, as failure to properly execute DSB repair is known to accelerate tumorigenesis and is associated with several human genetic syndromes. Here, we review recent insights into the mechanisms that influence the choice between competing DSB repair pathways, how this is regulated during the cell cycle, and how imbalances in this equilibrium result in genome instability.     

A genetic screen for DNA double-strand break repair mutations in Drosophila

The study of DNA double-strand break (DSB) repair has been greatly facilitated by the use of rare-cutting endonucleases, which induce a break precisely at their cut sites that can be strategically placed in the genome. We previously established such a system in Drosophila and showed that the yeast I-SceI enzyme cuts efficiently in Drosophila cells and those breaks are effectively repaired by conserved mechanisms. In this study, we determined the genetic requirements for the repair of this I-SceI-induced DSB in the germline. We show that Drosophila Rad51 and Rad54 are both required for homologous repair by gene conversion, but are dispensable for single-strand annealing repair. We provided evidence suggesting that Rad51 is more stringently required than Rad54 for intersister gene conversion. We uncovered a significant role of DNA ligase IV in nonhomologous end joining. We conducted a screen for candidate mutations affecting DSB repair and discovered novel mutations in genes that include mutagen sensitive 206, single-strand annealing reducer, and others. In addition, we demonstrated an intricate balance among different repair pathways in which the cell differentially utilizes repair mechanisms in response to both changes in the genomic environment surrounding the break and deficiencies in one or the other repair pathways.     

Roles of chromatin remodellers in DNA double strand break repair

Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways.     

New insights into the mechanism of homologous recombination in yeast

Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults. Repair of DSBs by homologous recombination provides an efficient and fruitful pathway to restore chromosomal integrity. Exciting new work in yeast has lately provided insights into this complex process. Many of the proteins involved in recombination have been isolated and the details of the repair mechanism are now being unraveled at the molecular level. In this review, we focus on recent studies which dissect the recombinational repair of a single broken chromosome. After DSB formation, a decision is made regarding the mechanism of repair (recombination or non-homologous end-joining). This decision is under genetic control. Once committed to the recombination pathway, the broken chromosomal ends are resected by a still unclear mechanism in which the DNA damage checkpoint protein Rad24 participates. At this stage several proteins are recruited to the broken ends, including Rad51p, Rad52p, Rad55p, Rad57p, and possibly Rad54p. A genomic search for homology ensues, followed by strand invasion, promoted by the Rad51 filament with the participation of Rad55p, Rad57p and Rad54p. DNA synthesis then takes place, restoring the resected ends. Crossing-over formation depends on the length of the homologous recombining sequences, and is usually counteracted by the activity of the mismatch repair system. Given the conservation of the repair mechanisms and genes throughout evolution, these studies have profound implications for other eukaryotic organisms.     

Capture of linear fragments at a double-strand break in yeast

Double-strand breaks (DSBs) are dangerous chromosomal lesions that must be efficiently repaired in order to avoid loss of genetic information or cell death. In all organisms studied to date, two different mechanisms are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Previous studies have shown that during DSB repair, non-homologous exogenous DNA (also termed ‘filler DNA’) can be incorporated at the site of a DSB. We have created a genetic system in the yeast Saccharomyces cerevisiae to study the mechanism of fragment capture. Our yeast strains carry recognition sites for the HO endonuclease at a unique chromosomal site, and plasmids in which a LEU2 gene is flanked by HO cut sites. Upon induction of the HO endonuclease, a linear extrachromosomal fragment is generated in each cell and its incorporation at the chromosomal DSB site can be genetically monitored. Our results show that linear fragments are captured at the repaired DSB site at frequencies of 10⁻⁶ to 10⁻⁴ per plated cell depending on strain background and specific end sequences. The mechanism of fragment capture depends on the NHEJ machinery, but only partially on the homologous recombination proteins. More than one fragment can be used during repair, by a mechanism that relies on the annealing of small complementary sequences. We present a model to explain the basis for fragment capture.     

Analysis of DNA double-strand break repair pathways in mice

During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.     

Dynamics of Chromatin during the Repair of DNA Double-Strand Breaks

Double strand breaks (DSBs) are arguably the most deleterious DNAlesion that a cell can sustain, and defects in the ability to detect and repair thesebreaks result in increased genomic instability and have been causatively linked tocancer. The repair of DNA DSBs must occur in the context of chromatin, andthere is increasing evidence that the modulation of chromatin plays an integralrole in the DNA DSB repair process. Here, we summarize a number of keyfindings, largely from studies performed in budding yeast, highlighting the role ofchromatin in DNA DSB responses.     

Molecular-genetic analysis of dual-stranded DNA break repair in saccharomyces yeasts

DNA double-strand breaks (DSB) are the most dangerous damage to genetic material caused by ionizing radiation and some chemical agents. Nonrestored DSB lead to chromosomal rearrangements, genetic instability, and cell death. On the other hand, DSB normally occur in cells in the course of normal gene functioning. DSB repair not only protects cells from adverse consequences and maintains stability of genetic material but is directly involved in the most important processes of cell life, such as meiosis and humoral immunity in vertebrates. The diverse mechanisms of homologous and nonhomologous recombination underlie DSB repair. In this respect, yeast are the best-studied object. In this review, genetic control and molecular models of the recombination DNA DSB repair in Saccharomyces cerevisiae are considered. Evidence has accumulated that indicates the higher eukaryotes retained the basic set of the repair pathways characteristic of bacteria and lower eukaryotes. However, different repair mechanisms predominate in yeast as compared to higher eukaryotes. Therefore, the results obtained in yeast experiments may be applicable to higher eukaryotes.     

Double-strand breaks repair by gene conversion in silkworm holocentric chromosomes

Maintenance of genome stability relies on the accurate repair of DNA double-strand breaks (DSBs) that arise during DNA replication or introduced by DNA-damaging agents. Failure to repair such breaks can lead to the introduction of mutations and chromosomal translocations. Several pathways, homologous recombination, single-strand annealing and nonhomologous end-joining, are known to repair DSBs. So far in the silkworm Bombyx mori, these repair pathways have been analyzed using extrachromosomal plasmids in vitro or in cultured cells. To elucidate the precise nature of the chromosomal DSB repair pathways in cultured silkworm cells, we developed a luciferase-based assay system for measuring the frequency of chromosomal homologous recombination and SSA. An I-SceI-induced DSB, within a nonfunctional luciferase gene, could be efficiently repaired by HR. Additionally, the continuous expression of the I-SceI endonuclease in the HR reporter cell allowed us to investigate the interrelationship between HR, SSA and NHEJ. In this study, we demonstrated that chromosome DSBs were mainly repaired by NHEJ and HR, whereas SSA was unlikely to be a dominant repair pathway in cultured silkworm cell. These results indicate that the assay system presented here will be useful to analyze the mechanisms of DSB repair in insect cells.     

Analysis of one-sided marker segregation patterns resulting from mammalian gene targeting

The double-strand break repair (DSBR) model is currently accepted as the paradigm for acts of double-strand break (DSB) repair that lead to crossing over between homologous sequences. The DSBR model predicts that asymmetric heteroduplex DNA (hDNA) will form on both sides of the DSB (two-sided events; 5:3/5:3 segregation). In contrast, in yeast and mammalian cells, a considerable fraction of recombinants are one sided: they display full conversion (6:2 segregation) or half-conversion (5:3 segregation) on one side of the DSB together with normal 4:4 segregation on the other side of the DSB. Two mechanisms have been proposed to account for these observations: (i) hDNA formation is restricted to one side of the DSB or the other, and (ii) recombination is initially two sided, but hDNA repair directed by Holliday junction cuts restores normal 4:4 segregation on that side of the DSB in which the mismatch is closest to the cut junction initiating repair. In this study, we exploited a well-characterized gene-targeting assay to test the predictions that these mechanisms make with respect to the frequency of recombinants displaying 4:4 marker segregation on one side of the DSB. Unexpectedly, the results do not support the predictions of either mechanism. We propose a derivation of mechanism (ii) in which the nicks arising from Holliday junction cleavage are not equivalent with respect to directing repair of adjacent hDNA, possibly as a result of asynchronous cleavage of the DSBR intermediate.     

Sex and the single (double-strand) break

It has been known for some time that DNA double-strand breaks (DSBs) initiate homologous recombination during meiosis. Two recent studies show that the fate of a single DSB in yeast is strongly influenced by the presence of other breaks in the genome, hinting that cell-wide or chromosome-regional mechanisms control the outcome of DSB repair.     

Rad52 has a role in the repair of sodium selenite-induced DNA damage in Saccharomyces cerevisiae

Selenium (Se) is a chemo-preventive agent that has been shown to have a protective role against cancer. The inorganic form of Se, sodium selenite (Na₂SeO₃), has frequently been included in various chemo-prevention studies, and this commercially available form of Se is used as dietary supplement by the public. Because high doses of this Se compound can be toxic, the underlying molecular mechanisms of sodium selenite toxicity need to be elucidated. Recently, we have reported that sodium selenite is acting as an oxidizing agent in the budding yeast Saccharomyces cerevisiae, producing oxidative damage to DNA. This pro-oxidative activity of sodium selenite likely accounted for the observed DNA double-strand breaks (DSB) and yeast cell death. In this study we determine the genetic factors that are responsible for repair of sodium selenite-induced DSB. We report that the Rad52 protein is indispensable for repairing sodium selenite-induced DSB, suggesting a fundamental role of homologous recombination (HR) in this repair process. These results provide the first evidence that HR may have a fundamental role in the repair of sodium selenite-induced toxic DNA lesions.     

Rad52 has a role in the repair of sodium selenite-induced DNA damage in Saccharomyces cerevisiae

Selenium (Se) is a chemo-preventive agent that has been shown to have a protective role against cancer. The inorganic form of Se, sodium selenite (Na2SeO3), has frequently been included in various chemo-prevention studies, and this commercially available form of Se is used as dietary supplement by the public. Because high doses of this Se compound can be toxic, the underlying molecular mechanisms of sodium selenite toxicity need to be elucidated. Recently, we have reported that sodium selenite is acting as an oxidizing agent in the budding yeast Saccharomyces cerevisiae, producing oxidative damage to DNA. This pro-oxidative activity of sodium selenite likely accounted for the observed DNA double-strand breaks (DSB) and yeast cell death. In this study we determine the genetic factors that are responsible for repair of sodium selenite-induced DSB. We report that the Rad52 protein is indispensable for repairing sodium selenite-induced DSB, suggesting a fundamental role of homologous recombination (HR) in this repair process. These results provide the first evidence that HR may have a fundamental role in the repair of sodium selenite-induced toxic DNA lesions.     

Equal sister chromatid exchange is a major mechanism of double-strand break repair in yeast

Equal sister chromatid exchange (SCE) has been thought to be an important mechanism of double-strand break (DSB) repair in eukaryotes, but this has never been proven due to the difficulty of distinguishing SCE products from parental molecules. To evaluate the biological relevance of equal SCE in DSB repair and to understand the underlying molecular mechanism, we developed recombination substrates for the analysis of DSB repair by SCE in yeast. In these substrates, most breaks are limited to one chromatid, allowing the intact sister chromatid to serve as the repair template; both equal and unequal SCE can be detected. We show that equal SCE is a major mechanism of DSB repair, is Rad51 dependent, and is stimulated by Rad59 and Mre11. Our work provides a physical analysis of mitotically occurring SCE in vivo and opens new perspectives for the study and understanding of DSB repair in eukaryotes.     

Poetry in motion: Increased chromosomal mobility after DNA damage

Double-strand breaks (DSBs) are among the most lethal DNA lesions, and a variety of pathways have evolved to manage their repair in a timely fashion. One such pathway is homologous recombination (HR), in which information from an undamaged donor site is used as a template for repair. Although many of the biochemical steps of HR are known, the physical movements of chromosomes that must underlie the pairing of homologous sequence during mitotic DSB repair have remained mysterious. Recently, several groups have begun to use a variety of genetic and cell biological tools to study this important question. These studies reveal that both damaged and undamaged loci increase the volume of the nuclear space that they explore after the formation of DSBs. This DSB-induced increase in chromosomal mobility is regulated by many of the same factors that are important during HR, such as ATR-dependent checkpoint activation and the recombinase Rad51, suggesting that this phenomenon may facilitate the search for homology. In this perspective, we review current research into the mobility of chromosomal loci during HR, as well as possible underlying mechanisms, and discuss the critical questions that remain to be answered. Although we focus primarily on recent studies in the budding yeast, Saccharomyces cerevisiae, examples of experiments performed in higher eukaryotes are also included, which reveal that increased mobility of damaged loci is a process conserved throughout evolution.